Bottom Line:
Instead, a variable amount of necrotic tissue formed.Although necrotic area correlated significantly with amount of vessels and the 2-step strategy had significantly more vessels than the 1-step strategy, no significant reduction of necrotic area was found.In conclusion, the animal model developed here represents a highly challenging situation, for which a suitable engineered bone graft with better prevascularization, better resorbability and higher osteogenicity has yet to be developed.

ABSTRACTThe gold standard treatment of large segmental bone defects is autologous bone transfer, which suffers from low availability and additional morbidity. Tissue engineered bone able to engraft orthotopically and a suitable animal model for pre-clinical testing are direly needed. This study aimed to evaluate engraftment of tissue-engineered bone with different prevascularization strategies in a novel segmental defect model in the rabbit humerus. Decellularized bone matrix (Tutobone) seeded with bone marrow mesenchymal stromal cells was used directly orthotopically or combined with a vessel and inserted immediately (1-step) or only after six weeks of subcutaneous "incubation" (2-step). After 12 weeks, histological and radiological assessment was performed. Variable callus formation was observed. No bone formation or remodeling of the graft through TRAP positive osteoclasts could be detected. Instead, a variable amount of necrotic tissue formed. Although necrotic area correlated significantly with amount of vessels and the 2-step strategy had significantly more vessels than the 1-step strategy, no significant reduction of necrotic area was found. In conclusion, the animal model developed here represents a highly challenging situation, for which a suitable engineered bone graft with better prevascularization, better resorbability and higher osteogenicity has yet to be developed.

ijms-16-12616-f001: (A) Amount of nucleated cells extracted per bone marrow biopsy; each dot represents one donor; (B) Percentage of colony forming units per nucleated cells extracted from bone marrow biopsy; (C) Silicon mold with scaffold during seeding; (D) Tetrazolium (MTT) staining showing the distribution of cells along the periphery after seeding: the top image is a midline section, and the bottom is the outside surface. Black bar represents 1 mm.

Mentions:
Cell isolation yields and percentage of colony forming units from rabbit bone marrow aspirates showed donor variability (Figure 1A,B). The monolayer expansion of isolated primary BMSCs required 25.1 ± 5.4 days to reach approximately two million cells. The seeding efficiency of BMSCs onto the scaffolds using a fibrin hydrogel and a silicon mold (Figure 1C) was always greater than 99%. The cells were found distributed along the periphery of the scaffolds (Figure 1D).

ijms-16-12616-f001: (A) Amount of nucleated cells extracted per bone marrow biopsy; each dot represents one donor; (B) Percentage of colony forming units per nucleated cells extracted from bone marrow biopsy; (C) Silicon mold with scaffold during seeding; (D) Tetrazolium (MTT) staining showing the distribution of cells along the periphery after seeding: the top image is a midline section, and the bottom is the outside surface. Black bar represents 1 mm.

Mentions:
Cell isolation yields and percentage of colony forming units from rabbit bone marrow aspirates showed donor variability (Figure 1A,B). The monolayer expansion of isolated primary BMSCs required 25.1 ± 5.4 days to reach approximately two million cells. The seeding efficiency of BMSCs onto the scaffolds using a fibrin hydrogel and a silicon mold (Figure 1C) was always greater than 99%. The cells were found distributed along the periphery of the scaffolds (Figure 1D).

Bottom Line:
Instead, a variable amount of necrotic tissue formed.Although necrotic area correlated significantly with amount of vessels and the 2-step strategy had significantly more vessels than the 1-step strategy, no significant reduction of necrotic area was found.In conclusion, the animal model developed here represents a highly challenging situation, for which a suitable engineered bone graft with better prevascularization, better resorbability and higher osteogenicity has yet to be developed.

ABSTRACTThe gold standard treatment of large segmental bone defects is autologous bone transfer, which suffers from low availability and additional morbidity. Tissue engineered bone able to engraft orthotopically and a suitable animal model for pre-clinical testing are direly needed. This study aimed to evaluate engraftment of tissue-engineered bone with different prevascularization strategies in a novel segmental defect model in the rabbit humerus. Decellularized bone matrix (Tutobone) seeded with bone marrow mesenchymal stromal cells was used directly orthotopically or combined with a vessel and inserted immediately (1-step) or only after six weeks of subcutaneous "incubation" (2-step). After 12 weeks, histological and radiological assessment was performed. Variable callus formation was observed. No bone formation or remodeling of the graft through TRAP positive osteoclasts could be detected. Instead, a variable amount of necrotic tissue formed. Although necrotic area correlated significantly with amount of vessels and the 2-step strategy had significantly more vessels than the 1-step strategy, no significant reduction of necrotic area was found. In conclusion, the animal model developed here represents a highly challenging situation, for which a suitable engineered bone graft with better prevascularization, better resorbability and higher osteogenicity has yet to be developed.